Phylogeography of the Crown-of-Thorns Starfish in the
Indian Ocean
Catherine Vogler1, John Benzie2, Paul H. Barber3, Mark V. Erdmann4, Ambariyanto5, Charles Sheppard6,
Kimberly Tenggardjaja7, Karin Gérard8, Gert Wörheide1,9*
1 Department für Geo- und Umweltwissenschaften and GeoBio-CenterLMU, Ludwig-Maximilians-Universität München, München, Germany, 2 Environmental Research
Institute, University College Cork, Cork, Ireland, 3 Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United
States of America, 4 Conservation International, Indonesia Marine Program, Bali, Indonesia, 5 Faculty of Fisheries and Marine Science, Diponegoro University, Semarang,
Indonesia, 6 Department of Biological Sciences, University of Warwick, Coventry, United Kingdom, 7 Ecology and Evolutionary Biology Department, University of California
Santa Cruz, Santa Cruz, California, United States of America, 8 Université de la Méditerranée, Station Marine d’Endoume, Marseille, France, 9 Bavarian State Collections of
Palaeontology and Geology, München, Germany
Abstract
Background: Understanding the limits and population dynamics of closely related sibling species in the marine realm is
particularly relevant in organisms that require management. The crown-of-thorns starfish Acanthaster planci, recently shown
to be a species complex of at least four closely related species, is a coral predator infamous for its outbreaks that have
devastated reefs throughout much of its Indo-Pacific distribution.
Methodology/Principal Findings: In this first Indian Ocean-wide genetic study of a marine organism we investigated the
genetic structure and inferred the paleohistory of the two Indian Ocean sister-species of Acanthaster planci using
mitochondrial DNA sequence analyses. We suggest that the first of two main diversification events led to the formation of a
Southern and Northern Indian Ocean sister-species in the late Pliocene-early Pleistocene. The second led to the formation of
two internal clades within each species around the onset of the last interglacial. The subsequent demographic history of the
two lineages strongly differed, the Southern Indian Ocean sister-species showing a signature of recent population
expansion and hardly any regional structure, whereas the Northern Indian Ocean sister-species apparently maintained a
constant size with highly differentiated regional groupings that were asymmetrically connected by gene flow.
Conclusions/Significance: Past and present surface circulation patterns in conjunction with ocean primary productivity
were identified as the processes most likely to have shaped the genetic structure between and within the two Indian Ocean
lineages. This knowledge will help to understand the biological or ecological differences of the two sibling species and
therefore aid in developing strategies to manage population outbreaks of this coral predator in the Indian Ocean.
Citation: Vogler C, Benzie J, Barber PH, Erdmann MV, Ambariyanto, et al. (2012) Phylogeography of the Crown-of-Thorns Starfish in the Indian Ocean. PLoS
ONE 7(8): e43499. doi:10.1371/journal.pone.0043499
Editor: Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, Canada
Received April 19, 2012; Accepted July 24, 2012; Published August 21, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was funded by the EU Marie-Curie Early Stage Research Training HOTSPOTS MEST-CT-2005-020561 and National Science Foundation (NSF)
grant OCE-0349177. GIS LAGMAY and Service des Pêches (Mayotte) gave financial support during field work. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have the following interest. Five Oceans LLC provided logistical support during the study. This does not alter the authors’
adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: woerheide@lmu.de
Understanding the extent of the genetic differences between
sister-species is especially important in organisms where the
presence of sibling species could have far-reaching impacts, such as
biological model organisms, commercially valuable species,
biological indicator species or organisms that require management, such as threatened species and pests [3,5]. The corallivorous
crown-of-thorns starfish (COTS) Acanthaster planci is of particular
interest in this regard as it undergoes population outbreaks that
have devastated coral reefs throughout much of its distribution
range since the 1960s [6]. Although outbreaks still account for a
large proportion of the disturbance to Indo-Pacific reefs today [7],
the causes of these outbreaks and appropriate monitoring
strategies to predict their occurrence and management plans to
reduce their impact are still debated [6–9].
Introduction
A growing body of research shows that cryptic speciation is
common in the marine realm (reviewed in [1,2]). Indeed,
molecular genetic surveys of natural populations are increasingly
identifying sibling species, closely related sister-species which are
often a priori morphologically indistinguishable and are thus
classified as a single nominal species [3]. This is even the case in
widespread marine organisms with long-lived pelagic larvae that
could be expected to display little genetic structure [1]. Identifying
closely related sibling species and the processes that drive their
speciation is essential to understanding evolutionary processes in
the marine environment and can shed light on the importance of
past and present barriers to gene flow in marine systems [4].
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Indian Ocean Acanthaster Phylogeography
called WNIO), and the other consisted of CR haplotypes found
only in the eastern and central northern Indian Ocean (ENIO;
Fig. 1). In the SIO sister-species, one clade consisted of CR
haplotypes found only in western Indian Ocean sites (WSIO), the
second consisted of CR haplotypes spread throughout the
southern Indian Ocean but apparently derived from ancestors
found in Cocos Keeling Islands, thus of eastern origin (ESIO;
Fig. 1). These clades and the central position of the Cocos Keeling
CR haplotypes were also recovered in the NeighborNets (Fig. S1),
supporting the robustness of this signal. The net divergence dA
between these clades was similar: 3.98% for WNIO vs. ENIO, and
3.50% for WSIO vs. ESIO, as were the TMRCAs for each lineage:
139’600 years ago for the NIO sister-species, 113’700 for the SIO
sister-species.
The Bayesian skyline plots showed some signs of recent
expansions in some populations of both sister-species, potentially
indicating an expansion after the last glacial maximum (18,000–
24,000 years ago), but in both cases a very large variance around
the parameter estimates limited the interpretability of the data
(Fig. S2). However, all other demographic statistics showed no
signs of a recent population expansion for the NIO sister-species
(Fs, D, and R2 not significant except Fs estimated with the CR
dataset; Table 1) whereas the SIO sister-species clearly did (Fs, D,
and R2 significant for both COI and CR; Table 1, see also Table 2
and 3).
Once thought to be a single species, research by Vogler et al.
[10] showed that the crown-of-thorns starfish is a species complex
comprised of four highly differentiated evolutionary lineages with
restricted ranges located in (i) the Pacific, (ii) the Red Sea, (iii) the
northern and (iv) the southern Indian Ocean. Phylogenetic
analysis indicates that the northern and southern Indian Ocean
clades are closely related sister groups, to the exclusion of the Red
Sea and Pacific clades, which also formed a clade, albeit with low
statistical support [10].
As an important and destructive predator on coral reefs, many
studies have examined the ecology and population dynamics in A.
plancii (e.g., [11,12,8,13,14]). However, the overwhelming majority
of COTS research has been performed on the Pacific species
under the assumption that these populations were representative of
the entire range. The failure to recognise the existence of a species
complex and extrapolation of Pacific COTS studies to the entire
distribution of COTS for both research and management
purposes may thus mask potentially important ecological differences among geographically unique lineages, contributing to a lack
of understanding of the processes that lead to regional outbreaks in
the different COTS lineages [10]. Indeed, although outbreaks are
also a reason for concern in the Indian Ocean [12,13] and the Red
Sea [15], they do not appear to be as massive or widespread as in
the Pacific [16], a pattern that might be indicative of key biological
or ecological differences between the sister-species.
Previous genetic studies on COTS populations have largely
focused on the genetic differences among Pacific and Indian
Ocean lineages, and have included limited geographic sampling
from the Indian Ocean [11,14,17]. In this study, we conduct a
basin-wide examination of COTS’ population genetic structure
within the Indian Ocean to 1.) identify the geographic distributions of the Northern and Southern Indian Ocean COTS lineages
and gain a better understanding of the processes that led to the
diversification of these two sister-species in the Indian Ocean, and
2.) explore differences in long-term population dynamics that may
have resulted from biological or ecological differences among the
two sibling species.
Spatial Genetic Structure and Migration Patterns
The overall WST of the NIO sister-species without a priori
structure was strong (WST = 0.51, p,0.001), whereas structure in
the SIO sister-species was weak (WST = 0.07, p,0.001). Indeed, 14
of the 36 pairwise WST comparisons in the NIO sister-species were
significant after Bonferroni correction, whereas none of the 55
SIO sister-species comparisons were (Table S4). There was
significant isolation by distance in the NIO sister-species as
revealed by the positive regression between WST/(12 WST) and the
logarithm of geographic distances (b = 1.28, R2 = 0.35, p,0.001;
Fig. S3a), and no relationship in the SIO sister-species (b = 0.11,
R2 = 0.06, p.0.05; Fig. S3b).
According to the AMOVA analyses, the regional groupings
explaining most of the genetic variation in the NIO sister-species
were composed of a western group (west: Oman and UAE), a
central group (central: Maldives) and an eastern group (east:
Thailand, Aceh, Christmas Island, Pulau Seribu, Krakatau and
Karimunjawa; Fig. 1). In the SIO sister-species, they followed the
Marine Ecoregions of the World provinces [19]: province 19
(prov19: Oman and UAE), province 20 (prov20: Kenya, Mayotte,
North Madagascar, South Madagascar, South Africa, Réunion
and Mauritius), province 22 (prov22: Chagos) and province 27
(prov27: Cocos Keeling Islands; Fig. 1). In the NIO sister-species,
most of the genetic variation was explained among regional groups
(57.37%, WCT = 0.574, p,0.001) within which variation was low
(2.82%, WSC = 0.066, p,0.001; Table 2). In the SIO sister-species,
although we present the regional combination that maximised
genetic variation among groups, this explained little of the total
variation (5.64%, WCT = 0.056, p,0.05), with most of the
variation occurring between individuals within populations
(92%; Table 2).
Migrate analyses based on the regional groupings identified by
the AMOVA were initially run with a full exchange matrix (i.e.,
bidirectional exchange of migrants possible between all regional
groups) to determine appropriate priors (Table S3). The process
was straightforward for the NIO sister-species, but the chains did
not converge for the SIO sister-species, even in very long runs.
Since the groupings in the SIO sister-species were of unequal sizes,
Results
Sampling and Sequencing
Of the 190 samples for which we obtained mitochondrial
putative control region (CR) sequences, 95 belonged to the
Northern Indian Ocean (NIO) sister-species (522 bp) and 95 to the
Southern Indian Ocean (SIO) sister-species (546 bp; Table 1,
Table S1). The corresponding mitochondrial partial cytochrome
oxidase subunit I gene (COI) dataset (632 bp) included 48
individuals of the NIO sister-species, and 57 of the SIO sisterspecies (Table 1, Table S1). Haplotype and nucleotide diversities
were high for the CR datasets, and lower for the COI dataset
(Table 1; see Table 2 and 3 for population level statistics). The
COI dataset was thus more appropriate for interspecific analyses,
and the CR datasets for intraspecific analyses.
Divergence Times and Demographic Patterns
The time of divergence between the two Indian Ocean sisterspecies was estimated to be 1.86–2.89 Mya, in the late Plioceneearly Pleistocene based on the net divergence dA of the K2P
distances from the COI dataset (divergence rate: 3.760.8%.Myr1; [18]).
Minimum spanning trees for each sister-species showed two
clades separated by a large internal split of 13 mutation steps. In
the NIO sister-species, one clade consisted of CR haplotypes found
only in the west and central northern Indian Ocean sites (here
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Indian Ocean Acanthaster Phylogeography
Table 1. Summary statistics per sister-species and dataset.
Dataset
Sequence length
(bp)
n
hD
P
FS
D
R2
Northern Indian Ocean sister-species
COI
632
48
0.68 (60.045)
0.004 (60.0059)
21.32
20.13
0.101
CR
522
95
0.98 (60.006)
0.020 (60.0102)
224.44
20.28
0.079
Southern Indian Ocean sister-species
COI
632
57
0.59 (60.074)
0.002 (60.0013)
212.67
22.08
0.036
CR
546
95
0.99 (60.003)
0.016 (60.0082)
224.65
21.57
0.050
COI: Cytochrome Oxidase I and CR: Control Region; bp, aligned sequence length; n, number of individuals; hD, haplotype diversity; p, nucleotide diversity; Fu’s FS;
Tajima’s D; Ramos-Onsins R2; significant values are bold.
doi:10.1371/journal.pone.0043499.t001
the shores of Indonesia to the Gulf of Oman, showed strong
genetic structure (WST = 0.51, p,0.001) between western (Oman
and UAE), central (Maldives) and eastern populations (Thailand,
Aceh, Christmas Island, Pulau Seribu, Krakatau and Karimunjawa) (Fig. 1). In contrast, in the Southern Indian Ocean sibling
species (Oman, UAE, Kenya, Mayotte, Madagascar, South Africa,
Réunion, Mauritius, Chagos, Cocos Keeling; Fig. 1), structure was
much weaker (WST = 0.07, p,0.001).
The recovery of distinct Indian Ocean lineages highlighted the
presence of barriers to genetic exchange within this ocean basin,
even though there are no obvious barriers to dispsersal and COTS
have relatively long pelagic larval durations of 3–4 weeks, based
on research from Pacific COTS [21]. That these two distinct
evolutionary lineages have radically different levels of genetic
structure across areas of the Indian Ocean without obvious
barriers to dispersal, despite having very similar geographic
ranges, strongly suggests that they are either impacted by different
environmental processes that shape connectivity and dispersal
across their range, or have unique ecological or biological
characters that influence their dispersal and connectivity. These
abiotic and biotic variables, either singly or in concert, then drive
differential evolutionary processes in the two species.
we restricted the analysis to the larger populations, i.e. within
prov20 only (excl. South Madagascar). However, the only model
that converged was the panmixia model, suggesting gene flow was
too high within this province to determine individual migration
rates between populations and allow a proper comparison of
migration models, a result consistent with the minimal CR genetic
structure in this species. Therefore, we only present the Migrate
results for the NIO sister-species where a series of different
migration models could be tested (Fig. 2).
Log Bayes factors indicate strong support [20] in favour of the
asymmetrical migration model M3, allowing migration from the
regional groups west and east towards central but not back to these
groups or between them (Table 4). For this model, the effective
number of migrants per generation (NeimjRi = Hi*MjRi) from west
to central was 218, and from east to central 254 (Table 5). The second
best ranking model, the full exchange model M1, essentially
revealed the same migration patterns as M3 but with a stronger
contribution to central’s gene pool from east than from west (Table 5).
Discussion
Previous genetic studies of Acanthaster planci have focused on
highlighting differences among Indian and Pacific Ocean populations using a limited number of Indian Ocean samples
[11,14,17]. However, increased sampling of the Indian Ocean
basin revealed the presence of sibling species within the Indian
Ocean [10], and rapidly evolving mtDNA control region sequence
data also indicates significant genetic structure within these sibling
species. The Northern Indian Ocean sister-species, ranging from
Diversification Processes
The application of a molecular clock suggests that the
diversification of the Northern (NIO) and the Southern Indian
Ocean (SIO) sister-species of the crown-of-thorns starfish occurred
during the late Pliocene-early Pleistocene (1.86–2.89 Mya).
Although the exact timing of this event should be interpreted
Table 2. AMOVA results for the Southern and Northern Indian Ocean sister-species.
Northern Indian Ocean sister-species
west vs. central vs. east
1
Southern Indian Ocean sister-species
prov19 vs. prov20 vs. prov22 vs. prov272
Overall WCT (between groups)
0.574***
0.056*
Overall WSC (within groups)
0.066***
0.025**
Among groups
57.37%
5.64%
Among populations within groups
2.82%
2.36%
Within populations
39.81%
92.00%
Percent variation:
1
west: UAE, Oman; central: Maldives; east: Thailand, Aceh, Christmas Island, Pulau Seribu, Krakatau, Karimunjawa.
prov19: UAE, Oman; prov20: Kenya, South Africa, Mayotte, South Madagascar, North Madagascar, Réunion, Mauritius; prov22: Chagos; prov27: Cocos Keeling Islands.
Significance tested with 50,000 permutations; *p,0.05, **p,0.01 and ***p,0.001.
doi:10.1371/journal.pone.0043499.t002
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Table 3. Summary statistics per location based on the Control Region dataset.
n
hT
hP
hF
h
p
FS
D
R2
UAE
2
2
1
0.50
1.00 (60.500)
0.004 (60.0045)
0.69
–
0.500
Oman
2
2
1
0.50
1.00 (60.500)
0.032 (60.0325)
2.83
–
0.500
Reunion
5
5
2
0.40
1.00 (60.127)
0.011 (60.0075)
21.06
20.28
0.138
Mauritius
4
4
4
1.00
1.00 (60.177)
0.010 (60.0074)
20.40
20.07
0.137
Kenya
24
22
17
0.77
0.99 (60.014)
0.017 (60.0090)
211.82
21.29
0.077
South Africa
12
12
11
0.92
1.00 (60.034)
0.017 (60.0094)
25.33
21.58
0.099
Mayotte
21
19
15
0.79
0.99 (60.018)
0.015 (60.0082)
29.55
21.17
0.081
Nth Madagascar
11
11
9
0.82
1.00 (60.039)
0.017 (60.0098)
24.40
21.38
0.091
Sth Madagascar
2
2
2
1.00
1.00 (60.500)
0.043 (60.0436)
3.14
–
0.500
Chagos
6
6
5
0.83
1.00 (60.096)
0.016 (60.0091)
21.23
21.14
0.055
Cocos Keeling Islands
6
3
2
0.67
0.73 (60.155)
0.003 (60.0024)
0.54
20.93
0.373
15
11
7
0.64
0.95 (60.040)
0.004 (60.0029)
25.67
20.60
0.084
0.123
Location
Southern Indian Ocean
sister-species
Northern Indian Ocean
sister-species
UAE
Oman
9
6
2
0.33
0.89 (60.091)
0.005 (60.0032)
21.66
20.77
Maldives
17
15
12
0.80
0.99 (60.025)
0.018 (60.0097)
25.51
0.07
0.119
Christmas Island
3
1
0
0.00
0.00 (60.000)
0.000 (60.0000)
–
–
–
0.093
Aceh
15
11
8
0.73
0.93 (60.054)
0.012 (60.0068)
22.48
21.13
Thailand
16
14
8
0.57
0.98 (60.028)
0.010 (60.0057)
27.55
20.45
0.116
Pulau Seribu
12
10
6
0.60
0.97 (60.044)
0.011 (60.0063)
23.21
20.49
0.122
Karimunjawa
5
4
3
0.75
0.90 (60.161)
0.012 (60.0083)
0.88
20.35
0.180
Krakatau
3
3
2
0.67
1.00 (60.272)
0.018 (60.0144)
1.07
–
0.205
Location
n
hT
hP
hF
hD
p
FS
D
R2
UAE
2
2
1
0.50
1.00 (60.500)
0.004 (60.0045)
0.69
–
0.500
Oman
2
2
1
0.50
1.00 (60.500)
0.032 (60.0325)
2.83
–
0.500
Reunion
5
5
2
0.40
1.00 (60.127)
0.011 (60.0075)
21.06
20.28
0.138
Mauritius
4
4
4
1.00
1.00 (60.177)
0.010 (60.0074)
20.40
20.07
0.137
Kenya
24
22
17
0.77
0.99 (60.014)
0.017 (60.0090)
211.82
21.29
0.077
0.099
Southern Indian Ocean
sister-species
South Africa
12
12
11
0.92
1.00 (60.034)
0.017 (60.0094)
25.33
21.58
Mayotte
21
19
15
0.79
0.99 (60.018)
0.015 (60.0082)
29.55
21.17
0.081
Nth Madagascar
11
11
9
0.82
1.00 (60.039)
0.017 (60.0098)
24.40
21.38
0.091
Sth Madagascar
2
2
2
1.00
1.00 (60.500)
0.043 (60.0436)
3.14
–
0.500
Chagos
6
6
5
0.83
1.00 (60.096)
0.016 (60.0091)
21.23
21.14
0.055
Cocos Keeling Islands
6
3
2
0.67
0.73 (60.155)
0.003 (60.0024)
0.54
20.93
0.373
UAE
15
11
7
0.64
0.95 (60.040)
0.004 (60.0029)
25.67
20.60
0.084
Oman
9
6
2
0.33
0.89 (60.091)
0.005 (60.0032)
21.66
20.77
0.123
Maldives
17
15
12
0.80
0.99 (60.025)
0.018 (60.0097)
25.51
0.07
0.119
Christmas Island
3
1
0
0.00
0.00 (60.000)
0.000 (60.0000)
–
–
–
0.093
Northern Indian Ocean
sister-species
Aceh
15
11
8
0.73
0.93 (60.054)
0.012 (60.0068)
22.48
21.13
Thailand
16
14
8
0.57
0.98 (60.028)
0.010 (60.0057)
27.55
20.45
0.116
Pulau Seribu
12
10
6
0.60
0.97 (60.044)
0.011 (60.0063)
23.21
20.49
0.122
Karimunjawa
5
4
3
0.75
0.90 (60.161)
0.012 (60.0083)
0.88
20.35
0.180
Krakatau
3
3
2
0.67
1.00 (60.272)
0.018 (60.0144)
1.07
2
0.205
n, number of individuals; hT, total number of haplotypes; hP, number of private haplotypes; hF, private haplotype frequency; hD, haplotype diversity; p, nucleotide
diversity; Fu’s FS; Tajima’s D; Ramos-Onsins R2; significant values are bold.
doi:10.1371/journal.pone.0043499.t003
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Indian Ocean Acanthaster Phylogeography
Figure 2. Migration models compared in the Migrate analysis
of the Northern Indian Ocean sister-species. Migration models
range from M1: full exchange to M6: panmixia. west (w), east (e) and
central (c) represent the regional groupings displayed in Fig. 1; arrows
indicate direction of migration.
doi:10.1371/journal.pone.0043499.g002
stands during glacial periods are thought to have restricted
dispersal pathways and/or altered the distribution of reef-dwelling
organisms [30], promoting evolutionary diversification. As NIO
and SIO COTS populations only known to overlap in the Gulf of
Oman, the most parsimonious hypothesis is that these lineages
diverged in allopatry. However, while sea level fluctuations are a
likely driver of divergence among the Pacific and Indian Ocean
COTS lineages [11], there are no emergent land barriers (such as
the Sunda and Sahul Shelves) in the Indian Ocean, indicating that
other processes must be driving diversification in this region.
The present distributions of the NIO and SIO sister-species are
largely, but not entirely, restricted to the two main current systems
to the north and south of the equator, respectively. The Indian
Ocean circulation is characterised by strong, seasonal monsoonal
current systems and upwelling patterns in the north, whereas an
equatorial gyre dominates the tropical southern half (Fig. 3; [31]).
The planktonic larvae of COTS display negative geotactic
behaviour, i.e. after hatching they swim to the surface and remain
there until the late brachiolaria stage (the last stage of their larval
cycle before settling; [21]). As such, ocean surface currents are
likely to have an important impact on their dispersal, and changes
in these currents can be expected to strongly affect the connectivity
between populations. It is therefore possible that the divergence of
the two species is based on these currents, as lowered sea levels of
the Plio-Pleistocene glacial periods are accompanied by pro-
Figure 1. Phylogeography of the crown-of-thorns starfish in
the Indian Ocean. (a) sampling locations from the Northern and
Southern Indian Ocean sister-species (here denoted as NIO and SIO
respectively), circles are proportional to sample size, colours indicate
the regional grouping of populations that explained most of the
variance amongst groups (NIO: w: west, c: central, e: east; SIO: prov19, 20,
22, 27 = Marine ecoregions regional provinces (Marine Ecoregions of
the World: http://www.worldwildlife.org/science/ecoregions/marine/
provinces.htm; [19])). (b) and (c) Minimum spanning trees (CR) of NIO
and SIO respectively, all haplotypes are separated by one mutational
step unless denoted by a higher number of hatch marks, except the
clades WNIO and ENIO as well a WSIO and ESIO which are separated by 13
mutational steps. Colours are the same as in (a) and circle size is
proportional to frequency of occurrence.
doi:10.1371/journal.pone.0043499.g001
Table 4. Performance of different gene flow models between
regional groupings in the Northern Indian Ocean sisterspecies (Fig. 2), ranked against M3, the best-performing
model.
with caution, as no external calibration points were available and
the mutation rate we used was inferred from other echinoderms
(Echinoidea; [18]), this general period coincides with periods of
strong climatically-induced sea-level fluctuations. Indeed, global
sea levels repeatedly dropped 120 m below their present level
during glaciations in the early Pleistocene (2.5, 2.2, 2.1 and 1.9
Mya; [22]).
Sea-level changes have frequently been invoked as a driver of
speciation on coral reefs [23,24], particularly among Pacific and
Indian Ocean populations (for reviews, see [25,26]), because the
dominant mode of speciation is allopatric [27,28] and there are
few obvious allopatric barriers in the sea [23,29]. Low sea-level
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Model
lM
LBF
Rank
M1
21954.69
213.4
2
M2
21975.56
234.3
3
M3
21941.29
0.0
1
M4
21995.19
253.9
5
M5
21991.02
249.7
4
M6
22031.98
290.7
6
lM: Log marginal likelihood, LBF: Log Bayes factors.
doi:10.1371/journal.pone.0043499.t004
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August 2012 | Volume 7 | Issue 8 | e43499
Indian Ocean Acanthaster Phylogeography
resulting from sea-level fluctuations may also have restricted the
distribution of COTS in the southern Indian Ocean. However, in
this area, past changes in circulation patterns are comparatively
poorly documented and still debated. Hutson [35] suggested that
intensified westerly winds would have hindered the penetration of
the South Equatorial Current and the Northeast Madagascar
Current along the southeast coast of Africa (Fig. 3), which could
have led to the retention of larvae between the continent and
Madagascar, and the subsequent diversification of these populations from other populations of the SIO sister-species. Although
more recent findings suggest that temperature and flow in this area
were stable for the last 150,000 years, changes in upwelling and
eddy formation may still have occurred [36]. The exact location of
divergences among the two southern Indian Ocean clades remains
unclear, although the central position of the Cocos Keeling
haplotypes in the minimum spanning tree might indicate that this
area possibly has acted as a refugium (prov27 in Fig. 1a), although
more data would be required to test this hypothesis.
The substantial evidence in favour of the impact of surface
circulation changes on population connectivity and subsequent
intraspecific divergence provides some support in favour of similar
dynamics having acted in the separation process of the two species.
However, at this point there is no evidence to suggest anything
more specific than that these currents might have helped to
maintain the isolation of these species following their divergence.
Comparative studies on a broad range of taxa in this region could
help clarify the processes driving diversification.
Table 5. Migration matrix of the two most supported gene
flow models in the Northern Indian Ocean sister-species (M3
and M1; Fig. 2).
from/to
west
west
0.015
654 (218)
0 (0)
0.010
178 (68)
23 (0.9)
central
east
central
east
0 (0)
0.333
0 (0)
60 (0.5)
0.379
77 (3)
0 (0)
762 (254)
0.015
56 (0.5)
693 (262)
0.038
Hi (diagonal) and the number of migrants from regional grouping i to j per
generation, followed by the migration rates in brackets. Top numbers are the
results for the asymmetrical model M3, bottom numbers for the full exchange
model M1.
doi:10.1371/journal.pone.0043499.t005
nounced changes in global climate that can have profound impacts
on ocean circulation.
Additional insights into the divergence of the Indian Ocean
COTS species can be gained from examining the divergence of
the major clades within each of these two sister-species. The close
timing of the intraspecific divergence (113–139,000 years ago) of
the two clades suggests these could have been initiated by one
single climatic event. Global sea levels also dropped 120 m below
their current level before the onset of the last interglacial
(30,000 years ago, isotopic stage 6; [32]). There is strong evidence
that during glacial periods, the northern Indian Ocean monsoonal
system would have been altered – the seasonal southwest (SW)
monsoon being weaker whereas the strength of the northeast (NE)
monsoon would have increased [33] in comparison to present-day
interglacial patterns (Fig. 3). As suggested by Pollock [34] when
investigating interspecific patterns of diversification in spiny
lobsters, weaker oceanic circulation could have increased the
retention of larvae in the Arabian Sea, thus promoting the
diversification of the west and east clades in the NIO sister-species.
In the SIO sister-species, we also detected a western and
eastern-origin clade (Fig. 3). Changes in surface circulation
Intraspecific Population Structure
Despite being closely related and ecologically similar sisterspecies, there were pronounced differences in the genetic structure
of the two COTS Indian Ocean lineages. The NIO sister-species
was characterized by strong genetic structure with three regional
groupings comprised of western, central and eastern Indian Ocean
populations (Table 2). An asymmetric pattern of connectivity was
detected between these regions where both western and eastern
populations feed into those of the central Indian Ocean (model M3
in Fig. 2, Table 5), suggesting that the latter is a dispersal sink. In
contrast, while the SIO sister-species has significant genetic
structure, it is much less pronounced in this species, suggesting
Figure 3. Schematic representation of the Indian Ocean surface circulation. (a) During the southwest (July/August) and (b) northeast
(December/January) monsoon after Schott and McCreary [67], in relation to crown-of-thorns starfish sampling locations (yellow circles: NIO sisterspecies, blue circles: SIO sister-species). Blue shaded areas indicate the area in which COTS larvae would likely be released according to season. Green
wedges in (a) are upwelling areas. Current branches indicated are the South Equatorial Current (SEC), Southeast and Northeast Madagascar Current
(SEMC and NEMC), East African Coast Current (EACC), Somali Current (SC), Ras al Hadd Jet (RHJ), West and East Indian Coast Current (WICC and EICC),
Southwest and Northeast Monsoon Current (SMC and NMC), South Java Current (SJC).
doi:10.1371/journal.pone.0043499.g003
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Indian Ocean Acanthaster Phylogeography
although larvae from the SIO sister-species are thought to be
released during the Austral summer [38]. However, high gene flow
was observed in the SIO sister-species, might suggest either
modern or (recent) past high connectivity even among extremely
isolated populations. Indeed, the Cocos Keeling Islands are
separated from their closest downstream neighbour, the Chagos
Archipelago, by 2700 km, and the latter from the Seychelles and
Rodrigues by another 1600 km. Travelling such large distances in
the open ocean far exceeds COTS pelagic larval duration in
normal conditions [21]. However, COTS larvae from the Pacific
sister-species have been found to extend their developmental
period to seven weeks in marginal food regimes [39], although the
occurrence of a facultative teleplanic larva remains to be
confirmed [6]. Productivity is generally much higher in the
northern Indian Ocean, with areas of high productivity
(.130 gC.m22) being distributed over a far greater proportion
of the northern Indian Ocean (generally associated with the
continental margins) than in the southern Indian Ocean (Fig. S5)
[40]. As such, low primary productivity in the southern Indian
Ocean might result in extended larval durations and higher
connectivity, consistent with our results of lowered levels of genetic
structure observed in the SIO sister-species, despite the greater
geographic distances among populations. In contrast, larval
duration in the northern Indian Ocean is unlikely to exceed that
found in normal conditions due to the high levels of primary
productivity, and we hypothesize that the resulting shorter larval
durations contribute to the stronger genetic structure observed in
the NIO sister-species.
The presence of a few individuals from the SIO sister-species in
populations of the NIO sister-species is quite intriguing (Oman;
Fig. 1a). These individuals do not appear to have dispersed into
the area during a single founder event, as their haplotypes do not
cluster together in the minimum spanning tree (Fig. 1c), suggesting
multiple dispersal and colonization events. As no individuals from
the SIO sister-species are found in the Maldives, the most likely
source of propagules would be the east African coast. Yet the
strong upwelling conditions and eddies that accompany the SW
monsoon (Fig. 3a) appear to be unsuitable for the transport of
larvae from this area to Oman [41]. During the NE monsoon,
when populations in the higher latitudes of the southern
Hemisphere are most likely to spawn, the southward flowing
Somali Current should also hamper the northward dispersal of
larvae (Fig. 3b). Although such oceanographic barriers to dispersal
should prevent larval crossing, it is clear that occasionally a few
propagules are transported against expectations [41]. As Glynn
[41] suggested for tropical species in this area, these may represent
ephemeral populations that experience brief periods of invasion
and extinction.
higher levels of connectivity across a similar geographic range.
Bayesian skyline plots indicate population expansion in both
species, suggesting non-equilibrium dynamics, although there was
a very large variance to those estimates. On the other hand, other
demographic statistics (Fu’s FS, Tajima’s D and Ramos-Onsins R2)
provide little support for non-equilibrium dynamics in the NIO,
while the SIO sister-species showed a strong signature of a recent
population expansion (Table 1). This suggests that the differences
in genetic structure among NIO and SIO populations may result
from regional differences in population stability, either a
consequence of abiotic or biotic causes. However, the extent of
the deep divergences (13 mutational steps, Fig. 1) separating W.
and E. Indian Ocean clades might represent a sampling bias, since
we lack samples, for example, from intermediate regions between
Kenya and Oman, i.e., the coasts of Somalia and Yemen.
Several hypotheses might explain the observed differences in
population genetic structure between the northern and southern
Indian Ocean. First, differences in landmass distribution may
impact patterns of connectivity. The northern Indian Ocean is
bounded by a long coastline on all but its southern margin, and
has large numbers of islands in the centre (Maldives) and east
(Andamans). While continuous coastline might be expected to
yield high genetic connectivity, there are major breaks in reef
distributions in the northern Indian Ocean. Upwelling areas off
Somalia and Oman, the northern Arabian Sea coast, stretches of
the western and eastern coast of Indian, and of the Bay of Bengal
lack coral reefs, creating potential barriers to dispersal. In contrast,
the southern Indian Ocean has extensive coastlines only on its
western and eastern reaches, but the only major breaks in coral
distributions are in southern Mozambique and in Madagascar,
which are close to the southern end of coral distribution [15] and
are therefore unlikely to have a major impact on connectivity in
COTS populations.
In addition to landmass impacting distribution of suitable
habitat, ocean currents may also play a significant role in creating
different levels of genetic structure in the two sister-species. As
described above, the main currents in the northern Indian Ocean
reverse according to monsoon, which along with strong changes in
upwelling patterns, leads to a complex current system. Although it
is untested whether data from the Pacific sister-species can be
extrapoalted to its other sister-species, COTS larvae are released
during a summer spawning season [6] at higher latitudes in the
Pacific Ocean (.10uN or S). This period corresponds to the SW
monsoon (Fig. 3a) in the northern Indian Ocean, when currents
potentially facilitate transport of larvae from the western to central
Indian Ocean, consistent with the analysis of gene flow (model M3
in Fig. 2). Although direct data on spawning times for populations
near the equator are rare, again, data from the Pacific Ocean if
extrapolated would suggest there is no discrete spawning season
[6]. Thus, movement of larvae from east to central Indian Ocean
might occur outside the SW monsoon, when currents flow from
east to west (Fig. 3b), with larvae from both the western and
eastern Southern Indian Ocean able to reach the Maldives with no
or few stepping-stones. Similarly, during the SW monsoon and NE
monsoon, the long pelagic larval duration (three to four weeks in
the Pacific; [21]) would enable larvae to travel 1200 km on the
predominant currents (Fig. S4; [37]), thus reaching the Maldives
from respectively Oman or Aceh either directly or within two
generations using a stepping-stone (e.g., western Indian coast or
Sri Lanka, respectively), resulting in higher connectivity in the
SIO.
In the southern Indian Ocean, the consistent gyre would
theoretically enable circulating larvae from east to west and viceversa throughout the year, independent of the spawning time,
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Conclusions
Although previously considered a single taxon, northern and
southern Indian Ocean populations of Acanthaster planci represent
genetically distinct sister-species. Differences in genetic structure
between them likely result from the interplay of ocean circulation
patterns, primary productivity, and proximity to land, all of which
combined impact the distribution of available habitat and larval
duration. While results clearly indicate that these species are on
different evolutionary trajectories, whether this differentiation has
led to changes in their biology requires further investigation. It is
conceivable that different selective pressures are acting on
individuals from the NIO and SIO sister-species, with longer
larval phases and better larval dispersal capabilities possibly being
selected for in the latter. As the general consensus today is that
outbreaks are at least to some extent caused by the effects of
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Indian Ocean Acanthaster Phylogeography
accurate COI divergence rates available for echinoderms to dA
(3.760.8%.Myr-1; [18]).
Intraspecific patterns of diversification were investigated by
estimating minimum spanning trees in Arlequin v3.5.1.2 [55] for
the CR sequences of both SIO and NIO, based on pairwise
differences and re-drawn with Adobe Illustrator. To assess the
robustness of the signal in the minimum spanning trees, we also
constructed split graphs in SplitsTree v4.11.3 [56] using the
NeighborNet method, which allow detecting incongruences in the
signal and alternative phylogenetic histories.
Because the minimum spanning trees revealed a deep internal
split, separating two clades in each species (Fig. 1), we estimated
the net divergence dA [53] between these clades using the CR
dataset (Table S1), as the COI sequences did not offer the
necessary resolution. After inferring the best-fit nucleotide
evolution model using the Akaike Information Criterion as
implemented in jModelTest v0.1.1 ([57]; TPM1uf+I+G for the
NIO sister-species, TrN+I+G for the SIO sister-species), dA was
estimated for the maximum likelihood distances calculated in
PAUP*4.0b10 [54].
Since there are no mutation rates available for echinoderm CR
sequences, we also used a concatenated COI-CR dataset to
calculate the time to the most recent common ancestor TMRCA of
both the NIO and SIO sister-species, by estimating Bayesian
skyline plots in BEAST v1.5.4 [58,59]. We set a strict clock on
COI since preliminary tests showed a clocklike behaviour of the
data could not be rejected (zero value of uncorrelated relaxed
lognormal clock standard deviation within 95% highest posterior
density interval). We used a substitution rate of 1.8560.4%.Myr21
(normal distribution) in order to incorporate the uncertainty on
this rate from the literature [18], and estimated the CR
uncorrelated relaxed lognormal clock from COI (see Table S2
for settings).
These Bayesian skyline analyses allowed us to explore the
demographic patterns within each of the sister-species, comparing
these to statistics that have the ability to detect signatures of recent
population expansions: Fu’s FS [60] and Tajima’s D [61], both
calculated using Arlequin v3.5.1.2 [55; 50,000 replicates], as well
as Ramos-Onzins R2 [62], estimated using the R package pegas
v0.3–1 ([63]; 10,000 replicates). All these demographic summary
statistics were estimated at the species level with the COI dataset,
and at the species, clade and population level with CR.
primary productivity on larval survival [6–8], such differential
selection could have far-reaching consequences for differences in
outbreak ecology between the Southern and Northern Indian
Ocean sister-species, a phenomenon that merits further investigation.
The results of this study also emphasize the importance of
conducting further genetic studies of coral reef-associated organisms in the Indian Ocean. There is very little population genetic
information available from this ocean [42], yet there is a strong
need for more research to increase the overall state of knowledge
[43] and devise appropriate conservation strategies [44,45]. By
identifying genetic breaks between and within species as well as
exploring the connectivity between populations [46,47], molecular
studies such as this one can not only increase our understanding of
the biology of individual organisms, but also contribute to
identifying conservation targets, and form the basis for biogeographical classifications and future monitoring [25,48].
Materials and Methods
Sampling and Sequencing
COTS samples were collected by SCUBA and snorkel from 18
sites in the Indian Ocean between 1990 and 2010 (Fig. 1, Table
S1). We excluded samples from the southeastern Indian Ocean
(Western Australia), as these populations have been previously
shown to belong to the Pacific sister-species [10]. We sampled
pyloric caeca [11], gonads [17] and/or tube feet. Tissue samples
were stored as soon as possible after collection, either at 280uC for
the pyloric caeca [11], or in ethanol (.80%), DMSO buffer [49]
and on FTA paper (Whatman) for the gonads and tube feet. The
DNA was extracted from the pyloric caeca using a MagAttract 96
DNA Plant Core Kit (Qiagen) according to the manufacturer’s
manual DNA purification protocol, with the following initial steps:
the tissue was manually ground in a 1.5 ml Eppendorf tube after
freezing in liquid nitrogen, then incubated at 35uC for an hour in
RLT lysis buffer (Qiagen), vortexed at full speed for 20 s, and
centrifuged at 80006g for 5 min. DNA was extracted from the
other tissues (gonads, and tube feet) using a DNeasy Tissue Kit
(Qiagen) according to the manufacturer’s protocol.
A DNA fragment containing the putative mitochondrial control
region (CR) and the 59 end of the adjacent 16S rRNA gene [50]
was amplified with the following primers: COTS-CR-F15635 59CAAAAGCTGACGGGTAAGCAA-39 and COTS-CR-R114 59TAAGGAAGTTTGCGACCTCGAT-39. DNA sequencing was
performed using the PCR reverse primer, and the following
internal forward primer: COTS-CR-seqIO-F15749 59GCTTGTGTTCACGGGAAAGC-39. Cytochrome Oxidase subunit I (COI) sequences from Vogler et al. [10] with additional
samples from the Chagos Archipelago (Table S1) were also used.
The sequences were assembled using CodonCode Aligner (http://
www.codoncode.com/aligner) and aligned in Seaview v4.2 [51]
using the built-in MUSCLE software [52]. All new sequences were
deposited in the EMBL nucleotide database (see Table S1 for
accession numbers).
Spatial Genetic Structure and Migration Patterns
All population level statistics were performed on the CR dataset
using Arlequin v3.5.1.2 [55], unless stated otherwise. We
calculated standard measures of genetic diversity (haplotype
frequencies, haplotype diversity hD and nucleotide diversity p)
for each population and sister-species (CR and COI), as well as
pairwise WSTs between population pairs within each sister-species
(50,000 random replicates, standard Bonferroni correction for
multiple tests). We also used a Mantel test (100,000 permutations)
to determine the relationship between genetic and geographic
distances within each sister-species following the method recommended by Rousset [64] for populations in a two-dimensional
model, i.e. testing the regression of population pairwise WST/(12
WST) against the natural logarithm of geographic distances [64].
We then used analyses of molecular variance (AMOVA) to identify
regional patterns of genetic differentiation (locus-by-locus
AMOVA, 50,000 replicates). We tested several different combinations of groups of populations. These groups were based on
geography and published regional provinces (Marine Ecoregions
of the World; [19]), with the aim to determine which combination
explained the most genetic variation among groups.
Divergence Times and Demographic Patterns
As the CR sequences could not be aligned unambiguously
between the Southern Indian Ocean (SIO) and the Northern
Indian Ocean (NIO) sister-species, the timing of their divergence
was estimated using the COI dataset (Table 1, Table S1). The net
divergence dA [53] between the two species was calculated using
Kimura 2-parameter (K2P) distances estimated in PAUP*4.0b10
[54], approximating divergence times by applying the most
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Indian Ocean Acanthaster Phylogeography
Figure S5 Areas of primary productivity higher than
130 gC/m22. In grey; modified from Reid et al., 2006, data for
1998–99 [not an El Niño year] after NASA SeaWiFS.
(PDF)
In order to understand the connectivity between the regional
groups identified by the AMOVA analyses (Table 2), we estimated
migration rates and effective population sizes with Migrate v3.1.6
[65], using the Control Region dataset and a Bayesian search
strategy as recommended by Beerli [66]. We established the most
likely mutation model available in Migrate by using PAUP*4.0b10
[54] to estimate parameters for site rate variation and the
transition/transversion ratio, and performed several exploratory
runs to determine appropriate priors (Table S3). To explicitly
evaluate the performance of different migration models, ranging
from panmixia to a full migration matrix (Fig. 2), we ran the
analyses with the following heating scheme: [1 1.5 3 10,000]
(1,000,000 generations, 32 replicates). This scheme allowed the
approximation of marginal likelihoods using thermodynamic
integration and hence the estimation of Bayes Factors to compare
the performance of different models [20].
Table S1 Sampling locations of crown-of-thorns starfish
individuals. With coordinates (decimal degrees), collector or
reference, number of Control Region (CR) sequences (nCR) and of
Cytochrome Oxidase I (COI) sequences (nCOI) per clade and
location, and EMBL accession numbers (in grey are EMBL
accession numbers from Vogler et al. (2008)). Locations preceded
by an asterisk are represented in both Indian Ocean sister-species,
locations preceded by a dash are shared with the Pacific sisterspecies.
(PDF)
Table S2 Run conditions for the BEAST Bayesian
Skyline analysis for both the Northern and the Southern
Indian Ocean sister-species.
(PDF)
Supporting Information
Figure S1 NeighborNet analyses of the (a) Northern and
(b) Southern Indian Ocean sister-species. The two main
clades within each species are highlighted, and the central Cocos
Keeling Island haplotypes in the ESIO clade are surrounded by a
grey box.
(PDF)
Table S3 Run conditions for the Migrate analyses
(Control Region dataset) for the Northern (NIO) and
Southern Indian Ocean (SIO) sister-species.
(PDF)
Table S4 Pairwise WST values for the (a) Northern and
(b) Southern Indian Ocean sister-species.
(PDF)
Figure S2 Bayesian skyline plots for the (a) Northern
and (b) Southern Indian Ocean sister-species. Black lines
are an estimate of effective population size as a function of time,
grey lines indicate the 95% upper and lower highest posterior
probability interval.
(PDF)
Acknowledgments
We acknowledge the Indonesian government for permits 1187/SU/KS/
2006 and 04239/SU.3/KS/2006, as well as the Oman Ministry of
Environment and Climate Affairs and the Fujeirah Municipality (UAE).
For logistic and/or financial support, thanks to GIS LAGMAY and Service
des Pêches (Mayotte), to Five Oceans LLC (Oman) and CORDIO
(Kenya). Support for Indonesian data collection came from the US
National Science Foundation (Biological Oceanography, OCE-0349177).
We also wish to thank Elisabeth Illidge-Evans, Gordon Kirkwood, Emre
Turak, Sven Uthicke and Shakil Visram for providing samples, as well as
all the institutions and individuals who supported the authors of this study
during fieldwork. We thank Oliver Voigt for helpful comments on the
manuscript.
Figure S3 Genetic distance WST/(12WST) as a function
of the natural logarithm of geographic distance (in km)
for the (a) Northern and (b) Southern Indian Ocean
sister-species.
(PDF)
Figure S4 Current direction and velocity during the
peak of (a) the Southwest Monsoon (January mean from
1993 to 2009) and (b) the Northeast Monsoon (July mean
from 1993 to 2009). Arrow colour indicates direction of flow
(westward: blue, eastward: red), arrow length and plot background
colour indicate current velocity in meters per second. Data
obtained from and plots constructed using Ocean Surface Current
Analysis – Real time: http://www.oscar.noaa.gov/(Bonjean and
Lagerloef 2002).
(PDF)
Author Contributions
Conceived and designed the experiments: CV JB GW. Performed the
experiments: CV. Analyzed the data: CV. Contributed reagents/
materials/analysis tools: CV JB PB A ME CS KT KG GW. Wrote the
paper: CV. Revised the manuscript: CV JB PB GW.
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August 2012 | Volume 7 | Issue 8 | e43499